Western blotting was conducted in accordance with previously described standard protocols [16 (link)]. Primary antibodies, including anti-Beta Actin (20536-1-AP, Proteintech, China), anti-α-SMA (14395-1-AP, Proteintech, China), anti-CD31 (11265-1-AP, Proteintech, China), anti-E-cadherin (20874-1-AP, Proteintech, China), anti-Vimentin (10366-1-AP, Proteintech, China), anti-ZEB1 (21544-1-AP, Proteintech, China), anti-HIF1α (20960-1-AP, Proteintech, China), anti-HIF1α-OH402 (ab72775, Abcam, USA), anti-HIF1α-OH564 (#3434, CST, USA), anti-PHD1 (12984-1-AP, Proteintech, China), anti-PHD2 (19886-1AP, Proteintech, China), and anti-PHD3 (18325-1-AP, Proteintech, China) were used. Secondary antibodies, consisting of goat anti-mouse and anti-rabbit antibodies conjugated with horseradish peroxidase (Proteintech, China), were utilized, and the blots were detected utilizing enhanced chemiluminescence (ECL) (P10300, NCM, China). Quantitative analysis of western blotting was performed using ImageJ.
Anti vimentin 10366 1 ap
Manufactured by Proteintech
Sourced in United States
Anti-Vimentin (10366-1-AP) is a primary antibody that targets the vimentin protein. Vimentin is an intermediate filament protein found in various cell types. This antibody can be used for the detection and analysis of vimentin expression in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti e cadherin 20874 1 ap, by Proteintech (4 mentions)
Anti gapdh 60004 1 ig, by Proteintech (3 mentions)
Anti e cadherin 60335 1 ig, by Proteintech (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Anti par1 26366 1 ap, by Proteintech (2 mentions)
Anti slug 12129 1 ap, by Proteintech (2 mentions)
Anti snai1 13099 1 ap, by Proteintech (2 mentions)
Tanon 5200multi device, by Merck Group (2 mentions)
Ab134045, by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Protease and phosphatase inhibitors, by Ipsen (2 mentions)
Ab92726, by Abcam (2 mentions)
Amersham hybond 0.45 m, by GE Healthcare (2 mentions)
Anti zeb 1 21544 1 ap, by Proteintech (2 mentions)
Hybond, by Cytiva (2 mentions)
Anti cd31 11265 1 ap, by Proteintech (1 mentions)
Anti beta actin 20536 1 ap, by Proteintech (1 mentions)
Anti α sma 14395 1 ap, by Proteintech (1 mentions)
Horseradish peroxidase, by Proteintech (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
10 protocols using anti vimentin 10366 1 ap
1
Comprehensive Western Blotting Protocol
2
Investigating miR-20-5p Regulation of RUNX3 and EMT
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Cells were seeded in 6-well plates with a density of 4 × 105 cells/well and were treated with miR-20-5p mimics and inhibitor for 48 h. Followed by treatment, cells were lysed with radioimmunoprecipitation assay (RIPA) Lysis buffer (Solarbio, Beijing, China) and the protein concentration was quantified using the bicincho-ninic acid (BCA) protein assay kit (Applygen Technologies, China). The simple western immunoblots were performed on a Wes (Protein Simple Santa Clara, San Jose, CA, USA) using the Size Separation Master Kit with Split Buffer (12– 230 D) according to the manufacturer's standard instruction. The antibodies used included the following: anti-RUNX3 (ab40278, abcam, UK), anti-Vimentin (10366-1-AP, Proteintech, USA), anti-E-cadherin (20874-1-AP, Proteintech, USA), and anti-GAPDH (10494-1-AP, Proteintech, USA).
3
Antibody Validation for Cell Analysis
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Anti-p53 (#2524S) and anti-Snail (#C15D3) were purchased from Cell Signaling Technology (Boston, USA), anti-CD31 (#ab28364), anti-VE-cadherin (#ab33168), anti-LC3 (#ab48394), anti-p62 (#ab56416), and anti-ATG5 (#ab108327) were obtained from Abcam (Cambridge, UK), anti-α-SMA (#A5228) was purchased from Sigma (St. Louis, USA), and anti-Vimentin (#10366-1-AP) was obtained from Proteintech (Wuhan, China).
4
Immunohistochemical Analysis of Protein Markers
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The immunohistochemical staining protocol has been described previously47 (link),50 (link),51 (link). In brief, paraffin-embedded tissue sections (5 μm) were immunostained with anti-DUSP2 (sc-32776, Santa Cruz), anti-E-cadherin (3195, Cell Signaling Technology), anti-N-cadherin (610920, BD Biosciences), anti-Vimentin (10366-1-AP, Proteintech), and anti-Phospho-ERK1/2 (4370, Cell Signaling Technology). The number of positive cells was counted in five randomly selected microscopic fields (×10, Nikon, Japan).
5
Protein Expression Analysis in Cell Lines
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Total protein extraction of cell lines was conducted using radio immunoprecipitation assay (RIPA) buffer and phosphatase inhibitors (Roche, Sigma, St. Louis MO, USA). Subsequently, we used the BCA protein concentration determination kit (Beyotime Institute of Biotechnology) to quantitate the protein concentration. Extracted proteins were resolved by SDS-PAGE and transferred to 0.22 μm PVDF membranes. The membranes were incubated with the corresponding primary antibody at 4 °C for 16 h, followed by secondary antibody with 1 h at room temperature. Finally, the protein bands were presented using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The relevant antibodies used for this study were antiNEK2 (sc55601, Santa Cruz Biotechnology), anti-E-cadherin (60335-1-Ig, Proteintech), anti-Vimentin (10366-1-AP, Proteintech), anti-N-cadherin (22018-1-AP, Proteintech), anti-MMP2 (ab97779, Abcam), anti-MMP9 (ab76003, Abcam), β-catenin (ab32572, Abcam), anti-cyclin D1 (1:200 dilution, ab16663, Abcam), anti- c-Myc (ab32072, Abcam), anti-GAPDH (60004-1-Ig, Proteintech).
6
Western Blot Analysis of Cell Signaling
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Cells were lysed with RIPA lysis buffer containing protease inhibitor cocktail (GenDEPOT, Harris County, TX, USA) for 20 min on ice. The lysate was centrifuged at 12,000 rpm for 30 min at 4°C. The protein concentrations were measured using a BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The lysates were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was incubated with 5% skim milk at room temperature for 1 h, then incubated with primary antibodies overnight at 4°C. After washing extensively, the membrane was incubated with HRP-conjugated secondary antibody for 2 h. The signal was detected using enhanced chemiluminescence system.
The primary antibody information used in this study is as follows: anti-KIF2A (ab197988), anti-GAPDH (ab8245), anti-N-cadherin (ab18203), anti-p-Akt (ab38449), anti-Akt (ab8805), anti-p-ERK (ab79483), anti-ERK (ab184699), anti-p-JNK (ab4821), anti-JNK (ab112501), anti-p-c-Jun (ab32385), and anti-c-Jun (ab40766) were purchased from Abcam; anti-E-cadherin (60335-1-Ig) and anti-Vimentin (10366-1-AP) were purchased from Proteintech.
The primary antibody information used in this study is as follows: anti-KIF2A (ab197988), anti-GAPDH (ab8245), anti-N-cadherin (ab18203), anti-p-Akt (ab38449), anti-Akt (ab8805), anti-p-ERK (ab79483), anti-ERK (ab184699), anti-p-JNK (ab4821), anti-JNK (ab112501), anti-p-c-Jun (ab32385), and anti-c-Jun (ab40766) were purchased from Abcam; anti-E-cadherin (60335-1-Ig) and anti-Vimentin (10366-1-AP) were purchased from Proteintech.
7
Gastric Cancer Protein Expression Analysis
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Immunohistochemical staining was performed to determine the protein expression of Mastl, Vimentin and epithelial (E-)cadherin in 126 gastric cancer tissues. Briefly, 4-µm 10% formalin-fixed (1 day at room temperature) paraffin embedded sections were dewaxed in xylene and antigen was retrieved by heating the sections (95°C for 30 min) in 10 mmol/l citrate solution (pH 6.0) for 30 min. After blocking the endogenous peroxidase activity, using 3% H2O2 (10 min at room temperature), anti-Mastl (#15739-1-AP, ProteinTech Group, Inc., Chicago, IL, USA), anti-Vimentin (#10366-1-AP, ProteinTech Group, Inc.) and anti-E-Cadherin (#20874-1-AP, ProteinTech Group, Inc.) (all dilution at 1:100) antibodies were incubated with the sections overnight at 4°C. Subsequently, sections were washed and treated with biotinylated horseradish peroxidase-labeled anti rabbit secondary antibody (#sc-2357, Santa Cruz Biotechnology, Inc., Dallas, TX, USA, 1:100) at 37°C for 30 min. Diaminobenzene was used to detect chromogen and hematoxylin was used as the nuclear counterstain at room temperature for 1 min, and staining was observed under light microscope (magnification, ×40). Non-specific rabbit immunoglobulin G (#sc-2027, at 1:100 dilution, Santa Cruz Biotechnology, Inc.) was used to replace the primary antibodies in immunohistostaining as negative controls.
8
Exosome protein profiling by Western blot
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Cell and exosome samples were lysed using RIPA buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (Epizyme, Shanghai, China). The protein concentration was quanti ed using a BCA protein assay kit (Beyotime). Proteins were separated by 10% (gradient) SDS-PAGE (Epizyme) and transferred onto PVDF membranes (Amersham Hybond 0.45µm; GE Healthcare). The membranes were blocked with 5% milk in trisbuffered saline-Tween (TBS-T) for 2h, then incubated with primary antibodies (anti-CD9(ab92726,Abcam),anti-CD63(ab134045,Abcam), anti-MMP-1(ab134184,Abcam), anti-PAR1(26366-1-AP,Proteintech),anti-Zeb-1(21544-1-AP,Proteintech), anti-Slug(12129-1-AP,Proteintech), anti-SNAI1(13099-1-AP,Proteintech), anti-E-cadherin(20874-1-AP,Proteintech), anti-Vimentin(10366-1-AP,Proteintech), anti-GAPDH(60004-1-Ig,Proteintech)) for 12h at 4°C. the membranes were washed with TBS-T for 3times,15min per time, then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at RT. The proteins were visualized using the ECL western blotting substrate (cat. WBKLS0100, Millipore) and a Tanon-5200Multi device.
9
Immunohistochemical Profiling of Cell Markers
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The immunohistochemistry (IHC) analysis was performed using the avidin biotin peroxidase complex (ABC) method as previously described17 (link). Anti-Ki67 (27309-1-AP) and anti-vimentin (10366-1-AP) rabbit polyclonal antibodies used in IHC experiments were obtained from Proteintech (Rosemont, IL).
10
Exosome protein profiling by Western blot
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Cell and exosome samples were lysed using RIPA buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (Epizyme, Shanghai, China). The protein concentration was quanti ed using a BCA protein assay kit (Beyotime). Proteins were separated by 10% (gradient) SDS-PAGE (Epizyme) and transferred onto PVDF membranes (Amersham Hybond 0.45µm; GE Healthcare). The membranes were blocked with 5% milk in trisbuffered saline-Tween (TBS-T) for 2h, then incubated with primary antibodies (anti-CD9(ab92726,Abcam),anti-CD63(ab134045,Abcam), anti-MMP-1(ab134184,Abcam), anti-PAR1(26366-1-AP,Proteintech),anti-Zeb-1(21544-1-AP,Proteintech), anti-Slug(12129-1-AP,Proteintech), anti-SNAI1(13099-1-AP,Proteintech), anti-E-cadherin(20874-1-AP,Proteintech), anti-Vimentin(10366-1-AP,Proteintech), anti-GAPDH(60004-1-Ig,Proteintech)) for 12h at 4°C. the membranes were washed with TBS-T for 3times,15min per time, then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at RT. The proteins were visualized using the ECL western blotting substrate (cat. WBKLS0100, Millipore) and a Tanon-5200Multi device.
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